The covalent attachment of a phosphopantetheinyl (4'-PP) arm to a variety of synthases and other proteins is a key posttranslational protein modification. The 4'-PP is installed on the proteins post-translationally from coenzyme A (CoA) on a conserved serine residue by action of phosphopantetheinyl transferase (PPTase) enzymes. Phosphopantetheinylation is essential for synthase activity, and more ..

The covalent attachment of a phosphopantetheinyl (4'-PP) arm to a variety of synthases and other proteins is a key posttranslational protein modification. The 4'-PP is installed on the proteins post-translationally from coenzyme A (CoA) on a conserved serine residue by action of phosphopantetheinyl transferase (PPTase) enzymes. Phosphopantetheinylation is essential for synthase activity, and removal of the PPTase gene precludes natural product synthesis in microorganisms, or in the case of fatty acid biosynthesis, renders the organism unviable. PPTase enzymes belong to a distinct structural superfamily. Within bacteria, these enzymes are grouped into two classes based upon primary structure, the AcpS-Type and Sfp-Type PPTases.

Sfp-type PPTases, corresponding to an activator of surfactin production in Bacillus subtilis, are responsible for modifying type I polyketide and nonribosomal peptide synthases of prokaryotes. Sfp-type PPTases are responsible for the activation of a variety of pathogen-associated virulence factors. Among these compounds are toxins such as mycolactone from Mycobacterium ulcerans, siderophores such as vibriobactin from Vibrio cholerae or mycobactin from Mycobacterium tuberculosis, as well as the mycolic acids which form the waxy cell wall of Mycobacteria. The biosyntheses of these natural products are considered attractive targets for drug design.

In search of small molecule Sfp-PPTase inhibitors, a fluorescence quenching assay was developed for detection of Bacillus subtilis Sfp-PPTase enzymatic activity in a miniaturized high-throughput format. The consensus ybbr acceptor peptide DALEFIASKLA was N-terminally labeled with Black Hole Quencher-2 (BHQ-2) and used in combination with rhodamine-labeled coenzyme A as a co-substrate. The PPTase-catalyzed reaction leads to a product containing both the rhodamine fluorophore and the BHQ-2 quencher covalently attached to the ybbr scaffold; thus, the rhodamine fluorescence, which in the starting state is unperturbed, is dramatically reduced upon its incorporation into the BHQ-2-tagged peptide.

Inhibition of Sfp-PPTase was detected by recording the fluorescence intensity change over a 30-minute reaction period at a 525 nm excitation and 598 nm emission. Reagents were supplied by Michael Burkart and Timothy Foley, University of California at San Diego.

3 uL of Sfp-PPTase enzyme (final concentration 15 nM) and 1 uL of substrate mixture (final concentration for rhodamine-CoA and BHQ-2-YbbR 5 uM and 12.5 uM, respectively) dispensed in columns 1, 2, 5 - 48. Columns 1 and 2 used to generate neutral control (uninhibited reaction) Assay Steps:Three uL of reagents, consisting of buffer (in columns 3 and 4 as negative control) and Sfp-PPTase (in columns 1, 2, 5 - 48) were dispensed into 1,536-well Greiner black solid bottom plate. Compounds (23 nL) were transferred via Kalypsys pintool equipped with 1,536-pin array. The plate was incubated for 15 min at room temperature, followed by the addition of 1 uL substrate to start the reaction. The plate was then centrifuged at 1000 rpm for 15 seconds, and the fluorescence intensity recorded on a ViewLux High-throughput CCD imager (Perkin-Elmer) using standard BODIPY optics (525 nm excitation and 598 nm emission). The plate was then incubated for 30 minutes, and a second read on the ViewLux was performed. The fluorescence intensity difference over the 30-minute period was used to calculate the respective reaction rate for each well.

Compound Ranking:1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.

Float

μM

3

Efficacy

Maximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.

Float

%

4

Analysis Comment

Annotation/notes on a particular compound's data or its analysis.

String

5

Curve_Description

A description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.

String

6

Fit_LogAC50

The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).

Efficacy at zero concentration of compound from a fit of the data to the Hill equation.

Float

%

11

Fit_CurveClass

Numerical encoding of curve description for the fitted Hill equation.

Float

12

Excluded_Points

Which dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.